The use of M-FISH to analysis the presence of genome instability in cells exposed to orthopaedic implant wear debris

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The use of M-FISH to analysis the presence of
genome instability in cells exposed to
orthopaedic implant wear debris

• Presented by Martin Figgitt
• Bristol Implant Research Centre
• Southmead Hospital
• Bristol

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Contents

• Introduction orthopaedic background, implant
  types etc

• The Bristol Study

• In vitro studies-metal ions

• Results

• Discussion

• Future work

• Acknowledgements

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Introduction

• Hip and knee orthopaedic implants the second
  most common elective operation in UK

• More orthopaedic implants in younger eg 40 years
  old

• Implants produce wear debris in both particulate
  and metal ion form ( eg cobalt and chromium)

• Does any of this wear debris have any detrimental
  effects upon patients eg genotoxicity

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The Bristol Study

• A comparison of the genotoxicity of two hip
  resurfacing implants Birmingham and ASR

• The study is comprised of three components

1)Patients M-FISH performed on pre-operative and
post-operative blood samples (6 months, 1 year
and 2 year)
2)In-vitro study of nanoparticle wear debris
3)In-vitro study of metal ions associated with
wear debris
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ASR
Birmingham
Hip Resurfacing Orthopaedic Implants
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Outline of procedure
X-ray-Implant in situ
Hip Resurfacing- Principle
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The metal ions in vitro model

• Use primary human fibroblasts (BJ)

• Expose cells to metal ions (Chromium, Cobalt)
  physiological concentrations

• Culture and harvest cells over a 30 day period

• M-FISH analysis (whole genome to be visualised)-
  to investigate genome instability

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Experimental Outline

• Fibroblasts (BJ cell line) seeded at 2.5x 105 per
  75 cm2 culture flask

• Cells left overnight to adhere, before exposure
  metal ions

• Pulse exposure to cobalt, chromium III and
  chromium VI ions

• One set of cultures exposed to the ions separately

• Second set with chromium III and chromium VI ions
  in combination with cobalt ions

• M-FISH analysis of fibroblast metaphases

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METAL ION EXPOSURE 24 HOURS
CoCl2
1.3,25,50 ppb
Cr III
HARVESTING AND PASSAGING
2,20,40 ppb
DAY 1,5,10,15---------30
Cr VI
2,20,40 ppb
Cr IIICo
1.3/2,25/20,50/40 ppb
Cr VICo
1.3/2,25/20,50/40 ppb
M-FISH ANALYSIS
Control
Experimental Schematic
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True Colour
Labelling Scheme
Pseudo Colours
M-FISH Metaphase
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Translocation
Deletion
Fragment
Aneuploidy
M-FISH Chromosomal Aberrations
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Chromosome aberration levels
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Do these aberration levels change overtime?
What is the composition of the aberrations, is
there a pattern?
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Summary

• Initial exposure to the metal ions induce
  chromosomal aberrations

• Over the time period analysis metal ion induced
  chromosomal aberrations regresses

• Both CrIII and Cr VI induce simple and complex
  aneuploidy

• The combination of Cobalt with CrIII and CrVI
  results in higher levels of chromosomal
  aberrations

• Cr VI displays a higher incidence and persistence
  of complex aneuploidy overtime with and without
  the presence of cobalt ions

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Future Work

• Compare metal ion results with nanoparticle
  experiments

• Investigate possible mechanisms causing
  aneuploidy eg tubulin disruption

• Prolonged exposure experiments

• Investigations with cell lines defective in DNA
  repair

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Acknowledgements
Dr CP Case Mr A Blom Professor I Learmonth Dr R
Newson
BIRC
Image Associates M-FISH technology
Grant Sponsors ARC
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Thank your for attention
Any questions?